Regulation of Tendon Repair with Stem Cell-Derived Extracellular Vesicles

项目摘要/摘要 肌腱和韧带损伤是最常见和最具挑战性的骨科疾病之一。很多病人 长期疼痛，受伤后功能减退。确凿的证据表明，这些 不满意的结果主要是由于过度和/或持续的不平衡的愈合反应造成的 炎症和肌腱再生不足。我们发现细胞外的囊泡是由 炎症诱导的脂肪干细胞(IEV)可能通过减毒来恢复平衡愈合 巨噬细胞介导的炎症和肌腱细胞驱动的刺激再生。但是，批量IEV 是异质的。差异产生的IEV携带不同的mRNA和microRNA分子，这些分子 确定IEV功能。有强有力的证据表明，只有一小部分IEV携带有活性分子 平衡巨噬细胞和肌腱细胞的损伤反应，而许多其他IEV可能 影响肌腱愈合或造成不想要的副作用。因此，有必要对IEV进行识别 肌腱修复特异的亚群和活性货物分子，并用它们来发展疾病特异性 治疗能大大加强肌腱修复。为了实现这一目标，批量IEV将按层次分类 基于确定泡囊货物的泡囊生成和货物分拣机器的有组织的子群 组成。专门促进肌腱愈合的亚群和活性货物分子将是 通过它们在调节肌腱细胞和巨噬细胞活动和功能方面的效力来鉴定。治疗性 已确定的IEV和CARO分子用于肌腱修复的有效性将在临床前小鼠跟腱上进行检验 肌腱损伤修复模型。我们预计移除功能性非特定IEV或选择性IEV 在原装IEV中浓缩功能特异的Cargo分子将大大提高治疗效果和 避免肌腱损伤治疗中大体积IEV的潜在副作用。这项研究是关键的证据-- 基于细胞外小泡(EV)治疗肌腱韧带临床应用的概念分析 伤害，这将使数百万患者受益。这项研究还将发现IEV的作用机制 肌腱损伤及肌腱细胞和巨噬细胞活动的分子调控机制 和功能。关于IEV的活性分子、表面标记和生物发生的知识 肌腱修复专用亚群将用于增加亲本对这些IEV的产量和释放 细胞，并通过基于免疫亲和力的方法放大IEV的生产。我们已经建立的方法可以用来 用于其他医疗条件下基于EV和疾病特异性治疗的未来发展，这将 改善更多患者的生活质量和预后。

PROJECT SUMMARY/ABSTRACT Tendon and ligament injuries are among the most common and challenging orthopedic conditions. Many patients suffer from long-term pain and reduced function after injury. Substantial evidence indicates that these unsatisfactory outcomes result primarily from an imbalanced healing response with excessive and/or sustained inflammation and inadequate tendon regeneration. We discovered that extracellular vesicles produced by inflammation-primed adipose-derived stem cells (iEV) could potentially restore balanced healing by attenuating inflammation mediated by macrophages and stimulating regeneration driven by tendon cells. However, bulk iEV are heterogeneous. Differentially generated iEV carry different mRNA and microRNA molecules, which determine iEV functions. There is strong evidence that only a fraction of iEV carry the active molecules that balance the injury responses of macrophages and tendon cells, whereas many other iEV could potentially compromise tendon healing or cause unwanted side effects. Therefore, it is necessary to identify the iEV subpopulation and active cargo molecules specific for tendon repair and use them to develop disease-specific therapy to greatly enhance tendon repair. To achieve this goal, bulk iEV will be sorted into hierarchically organized subgroups based on vesicle generation and cargo sorting machineries that determine vesicle cargo composition. The subpopulation and active cargo molecules that specifically enhance tendon healing will be identified by their potency in regulating tendon cell and macrophage activities and functions. The therapeutic efficacy of identified iEV and cargo molecules for tendon repair will be examined in a preclinical mouse Achilles tendon injury and repair model. We anticipate that removal of functionally non-specific iEV or selective enrichment of functionally specific cargo molecules in bulk iEV will greatly increase the therapeutic efficacy and avoid potential adverse side effects of bulk iEV for tendon injury treatments. This study is a critical proof-of- concept analysis toward clinical application of extracellular vesicle (EV)-based therapy for tendon and ligament injuries, which will benefit millions of patients. This study also will discover the mechanism of iEV action for tendon injuries, and mechanisms underlying the molecular regulation of tendon cell and macrophage activities and functions. Knowledge gained about the active molecules, surface markers, and biogenesis of the iEV subpopulation specific for tendon repair will be used to increase the yield and release of these iEV by parent cells and scale up iEV production via immunoaffinity-based method. Our established approaches can be used for the future development of EV-based and disease-specific therapies for other medical conditions, which will improve the quality of life and outcomes of more patients.